Apparatus and method for measuring the light absorbance of a substance in a solution

ABSTRACT

An apparatus for measuring the absorbance of a substance in a solution, includes at least one sample cell arranged to contain the solution that is at least partially transparent to light of a predefined wavelength spectrum, at least two light passages through the at least one sample cell, each of the light passages having a known path length, an LED light source arrangement including at least two LEDs, each arranged to emit a light output with a wavelength within the predefined wavelength spectrum. A plurality of optical fibers, one for each light passage, is arranged at each LED for receiving the light output and guiding it to the light passages. A method for measuring the absorbance of a substance in a solution includes providing the LED light source arrangement with an associate fiber bundle for each LED.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of PCT/EP2017/054504 filedon Feb. 27, 2017 which claims priority benefit of Great BritainApplication No. 1603380.5 filed Feb. 26, 2016. The entire contents ofwhich are hereby incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to an apparatus and method for measuringthe absorbance of a substance in a solution, typically a substanceexhibiting UV light absorption at a wavelength of 400 nm or less.

BACKGROUND

Many substances absorb ultra violet or visible light due to theirchemical composition. The absorption of light by substances has beenused as the basis for detecting the presence of, and measuring theconcentration of, such substances for many years. The concentration ofthe substance can be determined by use of the Beer Lambert Law:A=Ebc

where:

A is light absorbance;

E is the molar light absorptivity with units of L mol⁻¹ cm⁻¹;

b is the light path length of the sample defined in cm; and

c is the concentration of the compound in solution, expressed in mol⁻¹.

The UV region can be considered to consist of light of wavelength in theregion of 1 nm to 400 nm, light of wavelength of 180 nm to 300 nm beingknown as ‘deep UV’.

Most analytical instruments for detecting substances which absorb in thedeep ultra violet (UV) region use a mercury-lamp, deuterium lamp orxenon flash lamp as a light source. One example of such an instrument isa flow cell in which a solution containing one or more UV absorbingsubstances is passed between a UV light source (e.g. a mercury-lamp) anda UV detector (e.g. a photomultiplier or a photodiode) and changes inthe intensity of UV light reaching the detector are related to theconcentration of UV absorbing substances in the solution.

The detection of proteins, nucleic acids and peptides are of greatimportance in many sectors, including the environmental, biological andchemical sciences. Proteins have mainly two absorption peaks in the deepUV region, one very strong absorption band with a maximum at about 190nm, where peptide bonds absorb, and another less intense peak at about280 nm due to light absorption by aromatic amino acids (e.g. tyrosine,tryptophan and phenylalanine).

Nucleic acids absorb UV light at around 260 nm, some of the subunits ofnucleic acids (purines) having an absorbance maximum slightly below 260nm while others (pyrimidines) have a maximum slightly above 260 nm.Almost all proteins have a maximum absorbance at about 280 nm due to thecontent of the light absorbing aromatic amino acids. The light source inthe detectors of analytical systems used to detect and measure proteinconcentrations has historically been the mercury-line lamp. Mercuryproduces light with a wavelength of 254 nm but not at 280 nm, so afluorescence converter is needed to transform the 254 nm light producedby the mercury lamp to longer wavelengths and a band pass filter is usedto cut out a region around 280 nm. Mercury lamps have relatively shortlifetimes and can prove unstable with time; furthermore, the disposal ofthese lamps can lead to environmental problems. The other lamps used togenerate ultra violet light, such as the deuterium and the xenon flashlamps, disadvantageously require high voltages, need complicatedelectronics and often prove unstable with time. All of the currentlyused ultra violet light sources are relatively large and areconsequently unsuitable for miniaturisation of analytical instruments.Moreover, all of the lamps generate significant amounts of heat due tothe high voltages required for their operation.

Recently light emitting diodes (LED) of type AlGaN/GaN with emissions inthe 250 nm to 365 nm range have been developed. Sensor ElectronicTechnology, Inc. (Columbia, S.C., USA) have pioneered the developmentand use of these UV light 5 emitting diodes, particularly forirradiating and sterilising fluids such as biologically contaminatedwater (e.g. US 2005/0093485). Other groups have also employed UV lightemitting diodes for water purification systems (e.g. PhillipsElectronics, WO2005/031881).

Light emitting diodes (LEDs), which emit in the visible region of thespectrum, have been used for indirect photometric detection (Johns c.,et al. (2004) Electrophoresis, 25, 3145-3152) and fluorescence detectionof substances in capilliary electrophoresis (Tsai C., et al. (2003)Electrophoresis, 24, 3083-3088). King et al. (Analyst (2002) 127,1564-1567) have also reported the use of UV light-emitting diodes whichemit at 379.5 15 nm for indirect photometric detection of inorganicanions. The use of deep UV light emitting diodes as light sources indetection systems for nucleic acids is disclosed in US2005/0133724.However, although detection systems employing LEDs are disclosed, thereare no experimental data to indicate that the proposed systems wereindeed successfully employed to measure nucleic acid levels inpolymerase chain reaction assay. The system described would lacksensitivity, linearity, and dynamic range because there is no use of aband pass filter or a beam splitter and reference detector; LEDs arevery sensitive to minute changes in temperature, changes of the order ofone hundredth of a degree Centrigrade causing a drift in the baseline.Furthermore, the system lacks a band pass filter which acts to bothnarrow the bandwidth and block light in the visible region of thespectrum. A narrow bandwidth compared to the natural bandwidth of thesample, preferable a ratio of 1 to 10, provides a good linearity of theresponse and a broad dynamic range. (Practical Absorbance Spectrometry.Ed. A Knowles and C. Burgess, Chapman and Hall, New York).

JP2002005826 discloses a system for measuring ozone concentration.However, no experimental data that show the linearity and dynamic rangeare provided. The system uses a solid state emitter, which is composedof a diamond semiconductor thin film, to emit ultraviolet light with anemission peak of wavelength 240 to 270 nm. The emission spectrum at halfvalue width of the UV peak is somewhat narrower than the half valuewidth of the peak of the absorption spectrum of ozone (emission maximumapproximately 254 nm). However, while this may be sufficient to measureozone concentrations, the lack of a band pass filter which can reducethe band width to, for example, one tenth of the half value width of theozone absorption peak will significantly reduce the linearity anddynamic range of the detector (Practical Absorbance Spectrometry. Ed. AKnowles and C. Burgess, Chapman and Hall, New York). This system alsolacks a reference photo detector, so no measurement of the intensity ofthe emitted light is made. This means that compensation of variations ofthe emitted intensity due to changes in temperature is not possible.

WO2007/062800 (incorporated herein by reference), describes the use of aUV LED as a source of light for analysis of the concentration of asubstance in a liquid sample, but it has been found that a broaderspectrum of light is desirable in order to subject the sample todifferent wavelengths and thereby define a substance more accurately ormore quickly, by its absorption characteristics at differentwavelengths. However, known LEDs have only a limited light wavelengthoutput range.

WO2013/178770 discloses a system and method for measuring the absorbanceof a substance in a solution, using a plurality of LEDs with wavelengthwithin the UV spectrum to overcome the problems described above. Itwould however be advantageous to develop the technology further,especially with regard to cost efficiency and to create a more compactapparatus. The present invention addresses these problems to furtherimprove known methods and apparatuses within the field.

SUMMARY OF THE INVENTION

It will be understood that the term ‘substance’, as used herein, refersto any chemical entity. In particular, it includes organic compounds andinorganic compounds. Examples of organic compounds include, but are notlimited to, proteins, peptides, carbohydrates, lipids, nucleic acids,protein nucleic acids, drug candidates and xenobiotics. Examples ofinorganic compounds include metal salts (e.g. ferric sulphate, 30 copperchloride, nickel nitrate).

The object of the present invention is to eliminate or at least tominimize the problems described above. This is achieved through anapparatus and method according to the appended independent claims.

Thanks to the invention, the transmission of light from each of the LEDsto the light passages can be made more compact with fewer componentsthan previously known, resulting in a more efficient and cost effectiveapparatus. By arranging an associated optical fiber bundle, the bundlecomprising a plurality of optical fibers, the light output from each LEDcan be received by the bundle that provides a light guide from thatrespective LED to each of the light passages in the flow cell or flowcells without the need for additional components such as beam splittersand additional fiber bundles that have previously been required.

According to an aspect of the invention, a controller is provided tocontrol the light output from the light source arrangement. Thecontroller can select one of the LEDs to give the light output so thatthe light from only one of the LEDs reaches the light passages, but canalternatively also control the LEDs to each emit the light output atdifferent frequencies so that light from every LED reaches each lightpassage but generally not simultaneously.

The light passages may be arranged in the same flow cell but havingdifferent path lengths so that substances of different absorbance may beused with the invention. Alternatively, the light passages can bearranged in different flow cells each connected to the optical fibers ofthe apparatus.

The light output from the LEDs can be filtered to allow only onewavelength or a very narrow wavelength band of light to reach the lightpassages. The filter can be arranged between the LED and the opticalfibers, but can alternatively also be arranged between the optical fiberand the light passage or between the light passage and the detector. Inthe latter cases, one optical filter for each light passage and each LEDwill be required, for instance arranged on a filter wheel or similarstructure to allow for a removal and insertion of a specific filter whenthe corresponding LED is active. Another possibility is to provide afilter that allows light of wavelengths corresponding to more than oneof the LEDs, so that only one filter before or after each light passageis needed.

In one embodiment of the invention, the LEDs and optionally also theoptical filter arranged in connection with the LED may be replaceable sothat a user can select which wavelengths of light to use with theinvention.

Further advantages and benefits of the invention will become readilyapparent in view of the detailed description below.

DRAWINGS

The invention will now be described in more detail with reference to theappended drawings, wherein:

FIG. 1 discloses a schematic view of a preferred embodiment of theinvention;

FIG. 2 discloses a schematic view of another embodiment of theinvention;

FIG. 3 discloses a schematic view of a further embodiment of theinvention; and

FIG. 4 discloses a schematic view of yet another embodiment of theinvention.

DETAILED DESCRIPTION

FIG. 1 is a schematic representation of one embodiment of an apparatusaccording to the invention. The apparatus 10 comprises an LED lightsource arrangement 20 of light emitting diodes 21, 22, 23 which eachemit light in the ultraviolet part of the spectrum (UV LED) and two flowcells 31, 32, each having an inlet 313, 323 and an outlet 314, 324through which a solution containing a substance can pass in a flow F. Itis to be noted that in some applications, the light may have wavelengthsoutside the UV spectrum, such as visible light for instance, and thatwhat is said herein with reference to light within the UV spectrum alsoapplies to such applications using other LEDs.

The flow cells 31, 32 also comprise light passages 315, 325 with lightinlets 311, 321 and light outlets 312, 322 through which light from theLED light source arrangement 20 can pass and be received byphoto-detectors 41, 42, which can either be UV sensitive photomultipliers or UV sensitive photo diodes. The apparatus furthercomprises band pass filters 50 which reject unwanted wavelengths andadmits others, while maintaining a low coefficient of absorption for theUV wavelengths of interest. The bandwidth of the filter is a full widthhalf maximum, and is preferably less than 10 nm, to give a goodlinearity and large dynamic range.

This preferred embodiment comprises three LEDs 21, 22, 23 in the LEDlight source arrangement 20, each being arranged to emit light of aspecific wavelength within the spectrum that differs from thewavelengths of the other LEDs, and each being arranged with acorresponding optic filter 51, 52, 53 that is configured to allow thatspecific wavelength to pass while preventing other light frompenetrating the filter. Adjacent to each LED 21, 22, 23 of the lightsource arrangement 20 are optical fiber bundles 60 having plural opticalfibers that are arranged side by side to receive light that has passedthrough the filter 51, 52, 53 and providing a guiding light from arespective LED to each of the light passages 315, 325. The optical fiberbundles 60 are arranged so that one optical fiber from each LED 21, 22,23 is connected to each light passage 315, 325 so that in thisembodiment each of the three LEDs 21, 22, 23 emits light to two opticalfibers 60. In other embodiments the number of LEDs may vary as can thenumber of light passages, and it is to be understood that what isdescribed herein with reference to this preferred embodiment can easilybe adapted to suit such differences in configuration. Alternatively, theoptical filters 50 may be placed as filters 51 a, 52 a, 53 a, 51 b, 52b, and 53 b at the light inlets 311, 321, as shown in FIG. 3 , or asfilters 50 a and 50 b between exits of light passages 315, 325 anddetectors 41 and 42, as shown in FIG. 4 .

The filters 51, 52, 53 at the LED light source arrangement 20 may bearranged on a wheel or similar structure to allow for a change of thefilters as desired. Alternatively, filters that allow a plurality ofwavelength bands to pass may be used so that a single filter may allowlight from all the LEDs 21, 22, 23 to pass.

The flow cells 31, 32 have windows forming the light inlets 311, 321which are made from a UV transparent material such as sapphire, quartzor synthetic fused silica and is of a known path length. Othermaterials, such as polymers could be used. The solution is passedthrough the flow cells 31 and 32 via the inlets 313, 323 and the outlets314, 324, in the direction of arrows F, and may contain a substance witha light absorption at 300 nm or less e.g. a protein or nucleic acid. UVlight from the LED arrangement 20 is used to irradiate the solution S inthe flow cells 31, 32, the light entering the flow cells 31, 32 throughthe UV transparent windows 311, 321, as indicated by the dotted lines.Light passing through the solution and exiting the windows 312, 322 isthen detected by the photodetectors 41, 42. The light propagating fromthe light passages through the flow cells 31, 32 is detected andquantified to determine the absorbance of the substance in the solutionin the flow cell, as is well known within the art.

Similar to the embodiments disclosed by FIG. 2 and described below, thelight inlets 311, 321 and/or light outlets 314, 324 may comprise opticalfibers, glass rods or similar to allow the light to enter and exit theflow cells 31, 32, and to determine the path length of the lightpassages.

A controller 70 is connected to the LED light source arrangement 20 tocontrol the operation of the apparatus by selecting which of the LEDs21, 22, 23 is to emit light to irradiate the solution in the flow cells31, 32. The selection can be made by switching only one of the LEDs 21on or by blocking the others 22, 23 to prevent their light from reachingthe flow cells 31, 32. Alternatively, the LEDs 21, 22, 23 may be allowedto emit light simultaneously but at different frequencies. Thecontroller 70 is also connected to the detectors 41, 42 and able toreceive signals corresponding to a quantification of light that haspassed through the light passages 315, 325 without being absorbed by thesolution. These signals may be analyzed and stored by the controller 70or may be transmitted to a separate unit (not shown) for furtheranalyses, storage and display. The controller may also be configured tocontrol the flow of solution in the flow cells 31, 32, or alternativelythat control may be performed by the separate unit.

Once the absorption of the solution is measured, the concentration ofthe substance in the solution can then be determined by use of the BeerLambert Law where the molar absorbtivity E of the substance is alreadyknown. This can be done manually or using a computer or the controller70 provided. Alternatively, the concentration of the substance can bedetermined by use of a dose-response curve which has previously beenproduced for the substance of interest at a given wavelength e.g. 280nm, or multiple response curves which are generated at differentwavelengths can be used. Such determinations are made using a computervia a data link to the controller 70. In some applications, it is thechange in absorbance that is of interest, for example during separationof proteins in a chromatographic column, and so there is no need todetermine the concentration of the substance. In that case, the molarabsorptivity (E) need not be known. Using two frequencies of light alsoallows this change in absorbance to be more closely monitored when theabsorbance reaches a threshold where switching to a second less absorbedlight can give a better resolution of the rate of change of absorption,and consequently the approach of a maximum or minimum of concentrationvalues.

In this embodiment, the flow F through the flow cells 31, 32 can be inparallel or in series, but in either case the flow can be sequentiallyor synchronously monitored using different UV frequencies to provide agreater range of absorbance values as the concentration of the substancein solution changes. In a modification the two flow cells may havedifferent light path dimensions, thereby further enhancing the range ofthe apparatus. For example where a substance has a low absorbance at afirst frequency, then a long light path can be used, and where the samesubstance has a high absorbance at a second frequency, then a short pathlength can be used.

FIG. 2 discloses an alternative embodiment using a dual flow cell 30with two light passages 305, 305′ of different path lengths. The LEDlight source arrangement 20 has only two LEDs 21, 22, each emittinglight that passes through optical filters 51, 52 and is transmittedthrough optical fibers 60 in order to reach the flow cell 30. To providethe different path lengths, glass rods 80 are provided and inserted intothe flow cell 30 to create a first and second light passage 305, 305′where the first light passage 305 has a significantly smaller pathlength than the second path length 305′. In other respects, theembodiment of FIG. 2 corresponds to the preferred embodiment disclosedabove, and it is to be noted that features of these two embodiments mayfreely be combined.

The apparatus according to the invention may be made more cost effectivethan previously known devices, using fewer components and requiring lessspace than other known devices.

The LEDs of the light source arrangement 20 and their correspondingoptical filters 50 may be replaceable to allow for the substitution ofLEDs with light output of different wavelengths. This has the advantageof increasing the number of substances whose absorbance can be measuredby the apparatus and.

In operation, each the embodiments rely on a controller 70 to controlthe moment when the sample is irradiated. Since it is a straight forwardtask to alter the point in time at which the respective UV LED provideslight to the sample cell, and the apparatus employed is rugged and lowcost, then the embodiments shown provide an adaptable, reliable and lowcost liquid device for determining the concentration of a substance in aliquid by measuring its absorbance. It is preferred that UV LEDsemitting light up to 400 nm are used for the measurement ofconcentrations in solution of proteins, peptides, nucleic acids, cellextracts, cell lysates, cell cultures or combinations thereof, but theinvention has application to other light wavelengths, particularlywavelengths up to 700 nm. Two or three LEDs have been shown, but morethan three may be employed, for example four, or five or six or moreLEDs could be used, and additional LED's could emit visible light. Inthe embodiments, the band pass filters have been shown to be locatedbetween the sample cells 30, 31, 32 their respective LED light sources,however, the apparatus shown will function with equal effectiveness ifthe filters are placed after the sample cells, but before the detectors41, 42. In that case, the filters will need to be changed so that thecorrect filter is used with the correct LED.

The LEDs shown are schematically represented, and their form could bedifferent to that shown. So called multiple light source LEDs, whichgenerate different frequencies of light from adjacent semiconductorareas could be employed, in which case the scale of the devices shownwould be smaller, but there operating principles would be the same.

One mode of operation for all embodiments is to search for lowconcentrations of that substance at a first wavelength which substanceeven at low concentrations absorbs that light at the first frequencyreadily, and then, as concentrations increase, to switch to a secondwavelength which is not so readily absorbed, thereby providing a greaterrange of operation and sensitivity. In another mode of operation, LEDscan be powered in a predetermined cycle, and the output for the detectoris recorded in a matching cycle such that the light intensity from eachLED is recorded according to the cycle. Thereby, the output resultingfrom each LED can be determined because it is distinguished by adistinct set of values in a memory, corresponding to the cycle. Cyclingto differentiate between different LED's could be performed in time orfrequency domain. The cycle can be made very short in time, for examplefractions of a second (multiple Hertz), such that it appears to the eyethat the LEDs are illuminated simultaneously. The detector's supportingelectronics circuit can be arranged to inhibit or remove spurioussignals, for example by detecting output only during a predeterminedperiod within the switching cycle, to thereby remove noise from thesignal which might occur during the initial illumination or at the endof illumination, for a respective LED.

The apparatus and method according to the invention may also comprise areference detector arranged to receive a portion of light from the LEDto provide a reference signal for comparison with the signals from thedetectors. The light may be guided to the reference detector by means ofan optical fiber or may be guided in other suitable ways, and additionalcomponents such as beam splitters may be used to divide the light intoportions. In particular, where plural light guides are used to propagatelight to a flow cell, for example, different light guides for differentwavelengths of light from different sources, then it is envisaged that asingle reference detector, or a detector array could be used to receivelight from each of the sources, the light guides coming together at thedetector or at the detector array. In practice, it would be possible toroute one of plural optical fibers from each light source to each flowcell used, and another of the optical fibers to a reference detector, orto a detector array where multiple fibers are used, to provide areference value for each light source during its use. What is importantis the difference in light intensity measured by comparing the referencedetector intensity and the flow cell detector intensity rather than anany absolute valve of intensity, so the length of optical fiber used inthe reference path is not overly important, however for good practiceabout equal fiber lengths for the light guides used in the flow celllight paths and the reference detector light paths are preferred.

The above examples illustrate specific aspects of the present inventionand are not intended to limit the scope thereof in any respect andshould not be so construed. Those skilled in the art having the benefitof the teachings of the present invention as set forth above, can effectnumerous modifications thereto. These modifications are to be construedas being encompassed within the scope of the present invention as setforth in the appended claims. For determining the scope of thisdisclosure, it is intended that any feature of one embodiment could becombined with a further feature or features of one or more otherembodiments.

The invention claimed is:
 1. An apparatus for measuring the lightabsorbance of a substance in a solution, the apparatus comprising: atleast one sample cell arranged to contain said solution that is at leastpartially transparent to light of a predefined wavelength spectrum; atleast two light passages through a single sample cell of said at leastone sample cell, each of said light passages having a known path length;and an LED light source arrangement comprising at least two LEDs, eacharranged to emit a light output with a wavelength different to the otherLED or LEDs within said predefined wavelength spectrum, wherein each ofthe at least two LEDs includes an associated optical fiber bundle, theoptical fiber bundle comprising plural optical fibers, the optical fiberbundle providing a plurality of discrete light guides from a respectiveLED to each of the light passages, wherein the LED light sourcearrangement further comprises at least one optical filter for each LED,wherein said at least one optical filter for each LED is arrangedbetween the respective LED and the respective optical fiber bundle,wherein the at least two light passages in the single sample cell areassociated with different LEDs of the at least two LEDs, wherein the atleast two light passages are spaced apart from each other in the singlesample cell and wherein a total number of the at least one opticalfilter is less than a total number of the at least two LEDs.
 2. Theapparatus according to claim 1, further comprising a controller arrangedto control the light output from the LED light source arrangement. 3.The apparatus according to claim 1, wherein said at least one samplecell is a dual path length flow cell comprising said at least two lightpassages, and wherein each of said light passages has a path lengthdiffering from each of the other light passages.
 4. (PreviouslyPresented The apparatus according to claim 1, further comprising atleast two flow cells, each having at least one light passage.
 5. Theapparatus according to claim 1, wherein said at least one optical filtercomprises at least two optical filters for each LED, and wherein atleast one optical filter of the at least two optical filters is arrangedbetween an output end of a respective optical fiber of the pluraloptical fibers and an entrance of a corresponding light passage.
 6. Theapparatus according to claim 1, wherein said at least one optical filtercomprises at least two optical filters for each LED, and wherein atleast one optical filter of the at least two optical filters is arrangedbetween an exit of a respective light passage of the at least two lightpassages and a corresponding detector.
 7. The apparatus according toclaim 1, wherein at least one of the LEDs is arranged to be replaceable.8. The apparatus according to claim 1, including a reference detector ordetector array and wherein said optical fiber bundle includes an opticalfiber for providing a light path to said detector or detector array. 9.The apparatus according to claim 1, wherein the LED emits an ultravioletwavelength.
 10. The apparatus according to claim 1, wherein said samplecell has a flow inlet and a flow outlet configured to cause a sample toflow in a direction transverse to each of the at least two lightpassages.
 11. A method for measuring the light absorbance of a substancein a solution, comprising: providing an LED light source arrangementwith at least two LEDs, each arranged to emit a light output with awavelength different to the other LED or LEDs within a predefinedwavelength spectrum; providing for each LED an associated fiber bundle,the bundle comprising plural optical fibers, the bundle providing alight guide from a respective LED to at least two light passages throughthe substance in solution in a single cell; transmitting the lightoutput from each LED through at least one optical filter arrangedbetween the LED and the optical fiber; and quantifying the lightpropagating from the solution at each of said light passages to providean indication of the absorbance of the substance in the solution,wherein the at least two light passages in the single sample cell areassociated with different LEDs of the at least two LEDs, wherein the atleast two light passages are spaced apart from each other in the singlesample cell, and wherein a total number of the at least one opticalfilter is less than a total number of the at least two LEDs.
 12. Themethod according to claim 11, further comprising controlling the LEDlight source arrangement to select one of the LEDs for providing saidlight output.
 13. The method according to claim 11, wherein the at leastone optical filter comprises at least two optical filters, and whereinat least one optical filter of the at least two optical filters isarranged between the optical fiber and the light passage.
 14. The methodaccording to claim 11, wherein the at least two LEDs are caused to emitlight in a predetermined cycle and the quantifying step includesrecording the intensity of light propagating from the solution at eachlight passage an intervals corresponding to the cycle.
 15. The methodaccording to claim 11, wherein the step of providing an optical fiberbundle includes routing one or more of the optical fibers to arespective reference detector or detector array, and the step ofquantifying the light propagating from the solution, includes comparingthe light intensity propagating from the solution with the lightintensity at a respective detector or detector array.
 16. The methodaccording to claim 11, wherein the LED emits an ultraviolet wavelength.17. The method according to claim 11, further comprising flowing thesolution in a direction transverse to each of the at least two lightpassages.